Effects And Mechanisms Of The S24F RANTES/CCL5Variant On Cardiac Allograft Rejection

Part ⅠConstruction, identification and expression of recombinant adenovirus containing S24F RANTES/CCL5VariantObjective:With the aim of establishing the foundation for experimental research for intervention of transplant rejection in vivo and ex vivo, it is performed that the recombinant adenovirus vector containing S24F RANTES/CCL5variant gene is constructed and to investigate its expression for the gene therapy research.Methods:The nucleotide and amino acid sequences for the human RANTES/CCL5variant (S24F) have been published and used in the design and synthesis of the S24F gene. Resulting PCR products were cloned into the pAV-MCMV-GFP-3FLAG transfer vector at BamHI and EcoRI restriction sites. Recombinant plasmids were sequenced to confirm that the PCR product in the pAV-MCMV-GFP-3FLAG vector were in-frame following purification using the Wizard Plus SV Miniprep DNA Purification System. Recombinant adenoviruses were constructed using the AdMaxTM vector system. The pAV-MCMV/S24F-GFP-3FLAG and adenovirus DNA plasmid pBHGloxAE1,3Cre were co-transfected into HEK293cells. Cells were harvested after cytopathic effect was observed-and then subjected to3freeze-thaw cycles at-70℃ and37℃followed by two CsCl ultracentrifugation gradients to release recombinant viruses that had been stored in storage buffer. An adenoviral vector containing no transgene pAV-MCMV-GFP was used as a control. Virus titer was determined by Titer-EZ kit and S24F protein expression was determined by immunofluorescence, and Western blot.Results:Restriction endonuclease and PCR analysis demonstrated that the recombinant shuttle adenovirus vector pAV-MCMV/S24F-GFP-3FLAG was constructed correctly. The titer of Adeno-S24F-GFP-3FLAG (Ad-S24F) and Adeno-GFP (Ad-Null) stock preparations determined by the plaque titration assay were7.10×1011and2.00×1011plaque forming units (PFU)/mL, respectively. The results of immunofluorescence and Western blot showed that Ad-S24F was successfully constructed and can be expressed effectively.Conclusion:In this study, we successfully constructed a recombinant adenovirus vector that carries S24F RANTES/CCL5variant gene and can be expressed effectively in293T cells, which can be helpful for further research of the effects and mechanisms of the S24F gene on the transplant rejection. Part ⅡEffects of chemoattractant function of RANTES with adenovirus-mediated gene transfer of S24F of the human umbilical vein endothelial cellsObjective:To evaluate the expression of S24F with adenovirus-mediated gene transfer of S24F on human umbilical vein endothelial cells (HUVECs) in order to investigate the effects of chemoattractant function of RANTES in vitro.Methods:Isolation of human umbilical vein endothelial cells (HUVECs) was performed according to methods described by Jaffe et al. Transfection efficiency and expression of S24F in HUVECs preincubated with either Ad-S24F or the control adenovirus vector at a multiplicity of infection (MOI) of20,40, and60for48h was detected using the Cell Counting kit-8, fluorescence microscopy immunofluorescence, and Western blot analysis. The in vitro transendothelialchemotaxis assay was used to compare RANTES-induced transmigration of peripheral blood mononuclear cells (PBMCs) across HUVECs cultured on the upper Transwell chamber.Results:(1) Increasing MOI were associated with weak transfection efficiency and increased HUVEC mortality post transfection. MOI values below80did not alter HUVEC cell survival after transfection and an MOI of20resulted in the most efficient transfection rate (above95%).(2) HUVECs cultured for48h showed strong Flag-tagged S24F protein expression after infection with the adenoviral vector Ad-S24F by immunostaining with anti-Flag antibody, but not in HUVECs transduced with Ad-Null.(3) RANTES-induced PBMC transendothelialchemotaxis is inhibited by S24F.Conclusion:The constructed adenovirus (Ad-S24F and Ad-Null) can effectively mediate target gene expression in infected endothelial cells. Adenovirus vector could transfer target gene endothelial cells efficiently. Adenovirus-mediated gene transfer of S24F of the human umbilical vein endothelial cells may inhibit RANTES-induced PBMC transendothelialchemotaxis. Part ⅢThe effects and mechanisms underlying S24F gene transfer prolongs the survival of murine cardiac allograftObjective:To investigate the regulatory effect and underlying mechanisms of S24F gene on the survival time, inflammatory cell infiltration and associated proinflammatory cytokine expression profiles of cardiac allograft.Methods:The murine cardiac transplantation model was established (from BALB/C (H-2d) to C57BL/6J (H-2b)). After the donor heart was harvested and instilled slowly into the aortic root with500μL Dulbecco’s phosphate-buffered saline (DPBS) precooled to4℃. Subsequently,200μL of a solution containing Ad-S24F (1×109PFU), Ad-Null (1×109PFU), or PBS was perfused by intracoronary instillation ex vivo and subsequent incubation on ice for30min. Graft survival was assessed by performing daily abdominal palpation exams and the endpoint of the grafts defined as the absence of palpable contractions confirmed by direct laparotomy. Ninety mice were randomly divided into three groups:Ad-S24F transfected group (n=45), Ad-Null transfected group (n=25), and control group (n=20). The mice were sacrificed on days3,5,7,9,11, and14post-transplantation (4mice per time point). The time curve of expression of S24F in cardiac allograft and serum after advenovirus mediated transfer of S24F was detected by Western blot. The paraffined heart sections were stained with hematoxylin-eosin for routine cardiac pathohistology. Grafts were excised on day6post-transplant and immunohistochemical and integral optical density (IOD) analyses were used to estimate the presence of CD4+, CD8+, MOMA, and T-cell receptor (TCR) αβ+cells infiltrates in control or Ad-S24F transfected hearts. To better understand the inflammatory process associated with allograft transplantation we examined cardiac graft expression of various cytokine levels including RANTES, IFN-γ, IL-1β, TNF-α, TGF-β, CCR1, CCR3, CCR5, STATla, P-STAT1and IRF-1by Western blot and qRTPCR.Results:(1) Cardiac allografts transfection with Ad-S24F or Ad-Null showed intensive GFP fluorescence3days post transplantation. Expression of S24F was absent in allografts, and serum examined from Ad-Null group but GFP expression was observed. In the S24F transduced group, both S24F and GFP expression in allografts was detected. Peak S24F expression levels occurred between days3-5and decreased by day11. These results indicated that both S24F and GFP efficiently co-localized in allografts transfected with Ad-S24F.(2) Allograft survival in mice treated with Ad-Null (average survival9.38±0.60days) was similar to survival rates observed in PBS only treated mice (average survival9.00±0.38days, P>0.05). In contrast, allografts survival in mice treated with Ad-S24F were significantly prolonged (13.00±0.33days; P<0.05versus either control group).(3) Ad-S24F transduced allografts presented with significantly reduced CD8+lymphocyte, MOMA, and T-cell receptor (TCR) αβ+cell infiltrates (P<0.05). No significant reduction in CD4+lymphocyte levels was observed in the Ad-S24F group versus the Ad-Null group.(4) This analysis identified significant differences only in allograft protein expression and mRNA transcript levels of RANTES, IFN-y, CCR1, CCR3, CCR5, STATla, P-STAT1and IRF-1(P<0.05for each cytokine).Conclusion:RANTES/CCR and JAK/STATla signaling may be activated and S24F may play a role in pro-inflammatory signaling because the expression of RANTES, IFN-y, CCR1, CCR3, CCR5, STATla, P-STAT1and IRF-1was up-regulated after allograft transplantation in allografts of mice. However, local over-expression of S24F in the allograft can binds CCR1, CCR3, and CCR5causing receptor down-modulation and inhibits activation of JAK/STAT1α signaling by down-regulating pro-inflammatory cytokines expression and alleviating inflammatory response in murine cardiac allograft, suggesting that S24F may have a negative regulatory role in cardiac allograft rejection through binds CCR1, CCR3, and CCR5causing receptor down-modulation and negatively regulating the JAK/STATla signaling pathway. Thus, the RANTES/CCL5S24F natural mutant is an important component of the chemokine network involved in regulating the biological activity of RANTES.